Abstract

Addition of EGF to human carcinoma A-431 cells is known to induce membrane ruffling after approximately 2 min (Chinkers, M., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:260-265) and the phosphorylation of a protein referred to as p81, a known substrate for various protein-tyrosine kinases (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273). Ezrin, a Mr approximately 80,000 cytoskeletal protein of the isolated chicken microvillar core, is present in actin-containing cell surface structures of a wide variety of cells (Bretscher, A. 1983. J. Cell Biol. 97:425-432). Ezrin was then found to be homologous to p81 and to be phosphorylated on tyrosine in response to EGF (Gould, K. L., J. A. Cooper, A. Bretscher, and T. Hunter. 1986. J. Cell Biol. 102:660-669). Here, the purification of ezrin from human placenta is described. Antibodies to human ezrin, together with antibodies to other microfilament-associated proteins, were used to follow the distribution and phosphorylation of these proteins in A-431 cells after EGF treatment. EGF induces the formation of microvillar-like surface structures on these cells within 30 s and these give way to membrane ruffles at approximately 2-5 min after EGF addition; the cells then round up after approximately 10-20 min. Ezrin is recruited into the microvillar-like structures and the membrane ruffles, and is phosphorylated on tyrosine and serine in a time course that parallels the formation and disappearance of these surface structures. Spectrin is recruited into the membrane ruffles and shows a similar rapid kinetics of phosphorylation, but only on serine residues, and remains phosphorylated through the rounding up of the cells. The microvillar-like structures and membrane ruffles are also enriched in fimbrin and alpha-actinin. Myosin becomes rapidly reorganized into a striated pattern that is consistent with it playing a role in cell rounding. These results show that two cortical proteins, ezrin and spectrin, become phosphorylated in a time course coincident with remodeling of the cell surface. The results are consistent with the notion that ezrin phosphorylation may play a role in the formation of cell surface projections whereas spectrin phosphorylation may be involved in remodelling of more planar areas of the cell surface.